1. Field of the Invention
The present invention relates to chemical analytical systems and, more particularly, to such systems employing capillary electrophoresis to separate chemical sample components. A major objective of the present invention is to provide for convenient higher-resolution separations of sample components.
2. Description of the Related Art
Much of modern medical progress is due to advances in chemical analytical systems that permit an investigator to separate, identify and characterize various chemical moieties. A typical analytical procedure involves separating components of a sample mixture so that they can be individually identified and quantified, for example, using a form of spectroscopy.
Electrophoresis, like other classes of separation techniques such as chromatography, separates sample components by causing them to migrate at moiety-specific rates along a path. Electrophoresis applies an electric field to cause sample components to migrate at rates related to their charge-to-mobility ratios. As the different components separate, the molecules of each component diffuse so that single components form in bands. Each band can be characterized by a peak, where the component concentration is maximal, and a width, being the distance between points on either side of the peak where the concentration falls below some threshold criterion.
Roughly speaking, two sample components are resolved by a separation technique when their bands do not overlap. Accordingly, the resolving power of a separation technique is positively related with the peak separations it achieves and inversely related to the band spreading it incurs. To increase resolving power, peak separation must be increased proportionately relative to band width.
Due to diffusion, the resolution of a capillary electrophoresis system cannot be improved simply by increasing the length of the separation path. For example, if path length is doubled while the voltage differential across the path is held constant, then the electric field strength is halved. Migration velocities, which are proportional to field strength, are thus also halved. Transit times are quadrupled since distance is doubled and migration velocities are halved. Peak separations are doubled since, although time is quadrupled, the separating field is halved. Concomitantly, band widths, which expand proportionately with the square root of the transit time, are also doubled. Since peak separations and band widths are both doubled, there is no net gain in resolving power.
Increasing the voltage differential across the path can increase the resolving power of a capillary electrophoresis system. For a capillary of a given length, doubling the voltage differential doubles electric field strength. Doubling the electric field strength, in turn, doubles the migration rate and, thus, the differential migration rate. For a given length capillary, doubling the electric field causes separation to occur in half the time at twice the rate, so peak-to-peak separation is unchanged. Beneficially, however, diffusion is reduced by 2.sup.-1/2, resulting in a resolution increase of about 40%.
The extent to which resolution can be increased by increasing field strength is limited. The electric field causes the electrolytic medium through which the sample components migrate to heat. If the field is sufficiently strong, the resulting significant radial thermal gradients cause radial viscosity gradients. The viscosity of the electrolyte affects the migration rate of the sample components. The radial viscosity gradient thus results in a radial migration rate gradient, which in turn results in band broadening. Thus, reductions in band broadening due to diffusion can be partially or completely offset by band broadening due to radial migration rate gradients.
Accordingly, gains in resolution must be achieved by increasing voltage differentials without increasing field strengths beyond an optimal field strength range. This can be achieved by using longer capillaries. For example, for a given voltage differential, doubling the capillary length, halves the electric field strength. Diffusion is unchanged while band spreading due to migration rate differentials is reduced. Thus, a desired resolving power can be achieved by selecting a respective voltage differential and then selecting a capillary long enough so that electric field strength does not result in unacceptable migration rate gradients.
While the use of longer capillaries addresses the problem of excessive field strengths otherwise associated with greater voltage differentials, there are other more intractable problems with large voltage differentials. For example, voltage differentials of 30 kV are readily and economically achievable due to the prevalent use of 30 kV power supplies in the television industry. To achieve resolutions greater than those achievable using these readily available power supplies involves the use of much less economical alternatives. Moreover, the use of voltage differentials much above 30 kV incurs risks of arcing and corona.
Ancillary approaches to improving the resolution of a capillary electrophoresis system involve electro-osmotic flow control. Without such control, electro-osmotic flow is superimposed on the electrophoretic migration. The faster total flow decreases the time available for electrophoretic separation, thus decreasing the separation attainable for a given capillary length and separation voltage. Electro-osmotic flow can be reduced by column coating and applying sheath potentials. Each of these methods has its limitations: chemical specificity of coatings and pH-range limitations with sheath voltages.
Inevitably, no matter how great the resolving power of a capillary electrophoresis system, overlapping component bands will emerge. When overlapping bands occur, it would be desirable to resolve them further. However, in conventional electrophoresis systems, once sample components have traversed the separation path, there is no provision for further separation of the bands. Accordingly, what is needed is a system and method for achieving higher resolution systems without using excessive voltage differentials or field strengths. Preferably, such a system and method would permit increasing resolution through further processing.